The availability of complete genome sequences has created the demand for analytical tools to detect specific nucleic acid sequences (xNAs, which include DNA and RNA) in biological mixtures. These tools are needed in the clinic, in forensics, in exploring the microbial biosphere, as part of taggant detection systems, as well as in biomedical research laboratories. In the clinic, the tools are needed to detect low levels of single DNA/RNA targets that indicate the presence of infectious disease agents or biohazards. In both the research laboratory and the clinic, tools are needed to detect single nucleotide polymorphisms, as well as tools to detect multiple xNA targets at the same time on microarrays (in expression profiles, for example) [Pom02]. The purpose of the instant invention is to provide tools for doing this.
A general paradox is associated with architectures to detect specific nucleic acids in complex biological mixtures. First, as there are 4n different sequences of length n, a probe that is specific for a single sequence within a target genome having length G (nucleotides) will, on average, have a length L (nucleotides) that is given by the formula L=(log G)/0.6. Thus, for the human genome with ca. 3×109 nucleotides, a probe that is 16 nucleotides long will bind, on average, once to the target genome.
This type of calculation suggests that one can seek specific genes in a human genome using 16mer complements as probes. Unfortunately, for duplexes of this length under standard hybridization conditions, single mismatches depress the melting temperatures only slightly. Further, the different intrinsic affinities of the AT and GC nucleobase pairs, as well as the fact that the contribution of any nucleobase pair has a non-negligible dependence on the local sequence context, means that a duplex built from two 16mers having two mismatches can easily be more stable than a duplex built from two perfectly matched 16mers, even if they have the same overall GC/AT composition.
Of course, if the probe is shorter, then a duplex with a single mismatch will be less stable than any perfectly matched duplex. For DNA-DNA duplexes under standard conditions, this is certainly met by duplexes as short as 4 nucleobase pairs, and generally by duplexes as long as 10 nucleobase pairs. These, however, lack specificity in the human genome (a 10mer is found on average 4000 times in the human genome). Further, very short nucleotides (e.g. 4mers) are too short to truly display a “melting temperature”, a sharp transition between bound and unbound states as a function of temperature. Rather, the unbinding curves have the sigmoidal shape characteristic for a binary dissociation process.
Reversible template ligation can offer a solution to this problem. This approach differs from the irreversible template ligation suggested by Kool [San04], von Kiedrowski [Sie94] [Bag96] Templates, autocatalysis and molecular replication. Pure Appl. Chem., 68, 2145-2152. Ellington [Jam99] and others, in that the reversible ligation permits the system to achieve the thermodynamically preferred combination without kinetic traps.
Work by Lynn and his coworkers provides another part of the background for the instant invention [Goo92][Goo92][Zha97][Zha01]. In 1997, Lynn and his coworkers showed that complementary oligonucleotides could be assembled using imine chemistry from fragments under conditions of dynamic equilibrium [Yun97]. They did not propose this to be done in a combinatorial sense. Rather, their goal was to model chemistry that might create artificial replicating systems, themselves models for how life might have emerged on early Earth. Thus, Lynn and his coworkers used only short trinucleotides as their fragments, and immediately captured the imine by reducing it with borohydride to give a hexamer with a central, unnatural, CH2—CH2—NH—CH2 linker (FIG. 3). This created a stable secondary amine linker. They did not attempt to learn whether the imine formed transiently could serve as a primer.
Because Lynn and his group had the imine only transiently in hand, they could obtain only and approximate estimated the increased affinity due to the templating reaction, about a factor of 10. Further, they did not examine the infidelity of the process, the success of the process as a function of the length of the 3′- and 5′-fragments, or the fidelity of the process as a function of the length of the fragments. All are expected to be interrelated, and depend on the temperature, which will in turn be determined by the polymerase that is used to extend the fragment. This temperature must be low if reverse transcriptase is used to extend the primers (as in RNA transcription profiling, for example) or high if a thermostable DNA polymerase is used.
Of somewhat greater concern was the observation by Lynn and his coworkers that the binding of the secondary amine product obtained by the reduction of the imine to complementary DNA was lower than the binding of the analogous DNA-DNA complement [Luo98]. These authors examined the destabilization that arises from a single CH2—CH2—NH—CH2 linker in a short oligonucleotide, which can drop Tm as much as by 15° C. [Luo98]. From our work in this area [Hut02][Ben04], it is likely that the destabilization is due to the increased flexibility and complementary charge of the CH2—CH2—NH—CH2 unit. If so, the destabilization should be less with the imine CH2—CH═NH—CH2 linker.